EP2743684B1 - Method for detecting fluorescent particles - Google Patents

Method for detecting fluorescent particles Download PDF

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Publication number
EP2743684B1
EP2743684B1 EP12824491.0A EP12824491A EP2743684B1 EP 2743684 B1 EP2743684 B1 EP 2743684B1 EP 12824491 A EP12824491 A EP 12824491A EP 2743684 B1 EP2743684 B1 EP 2743684B1
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Prior art keywords
fluorescent
particles
sample solution
particle
detecting
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German (de)
English (en)
French (fr)
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EP2743684A4 (en
EP2743684A1 (en
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Hidetaka Nakata
Tetsuya Tanabe
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Olympus Corp
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Olympus Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6419—Excitation at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates

Definitions

  • the present invention relates to a method for detecting a fluorescent particle using an optical system capable of detecting light from a microregion in a solution, such as an optical system of a confocal microscope and multi-photon microscope.
  • fluorescence intensity is measured from fluorescent molecules or fluorescent-labeled molecules entering and leaving a microregion in a sample solution using the optical system of a laser confocal microscope and photon counting technology.
  • the average retention time (transitional diffusion time) of fluorescent molecules and the like in a microregion and the average value of the number of molecules remaining therein are determined from the value of an autocorrelation function of the measured fluorescence intensity.
  • the aforementioned microregion refers to a confocal region where laser light of a microscope is focused, and is also referred to as confocal volume.
  • a histogram is generated of the fluorescence intensity of fluorescent molecules and the like entering and leaving a measured confocal volume in the same manner as FCS, and by fitting a statistical model formula to the distribution of that histogram, the average value of the characteristic brightness of the fluorescent molecules and the like and the average value of the number of molecules remaining in the confocal volume are calculated. Changes in molecular structure or size, coupling and/or dissociation, dispersion or aggregation and the like are then estimated based on this information.
  • Patent Documents 5 and 6 propose a method for detecting a fluorescent substance based on the time lapse of a fluorescent signal of a sample solution measured using the optical system of a confocal microscope.
  • Patent Document 7 proposes a signal arithmetic processing technology for detecting the presence of fluorescent fine particles in a flow or on a substrate by measuring feint light from fluorescent fine particles that have passed through a flow cytometer or fluorescent fine particles immobilized on a substrate using photon counting technology.
  • the sample required for measurement is only required to be at an extremely low concentration and extremely small amount (since the amount used for a single measurement is roughly only several tens of microliters) and measurement time is shortened considerably (measurement of a duration on the order of several seconds for a single measurement is repeated several times) in comparison with that used in the past.
  • these technologies are expected to be utilized as powerful tools that make it possible to carry out experimentation or testing less expensively and faster in comparison with conventional biochemical methods in the case of performing analyses on scarce or expensive samples frequently used in fields such as medical or biochemical research and development, or in the case of a large number of specimens such as when clinically diagnosing diseases or screening physiologically active substances.
  • Patent Document 8 teaches that, if an amount of photons detected is greater than a threshold level, the presence of a label is detected.
  • the concentration or number density of fluorescent molecules and the like targeted for observation in a sample solution is preferably adjusted so that the number of fluorescent molecules and the like able to be statistically processed enter and leave a microregion within the duration of a single measurement having a length on the order of several seconds.
  • the concentration or number density of fluorescent molecules and the like targeted for observation in a sample solution is adjusted so that roughly only one fluorescent molecule and the like is present in the microregion at all times in an equilibrated state.
  • the concentration of fluorescent molecules and the like is preferably about 1 nM or more since the volume of the confocal volume is about 1 fL.
  • Patent Document 7 is a technology for individually detecting the presence of fluorescent fine particles flowing through a flow cytometer or immobilized on a substrate.
  • the technology described in Patent Document 7 is not a technology for detecting molecules or colloidal particles and the like dissolved or dispersed in an ordinary state in a sample solution, namely particles randomly moving in a sample solution.
  • Patent Document 7 since the technology of Patent Document 7 includes a process consisting of measuring in a flow cytometer or immobilizing fluorescent particles on a substrate, the amount of sample required for testing is much larger in comparison with the case of optical analysis technologies such as FCS, FIDA or PCH, and is thought to require the person performing testing to perform a complex and sophisticated procedure.
  • an embodiment of the present invention does not include statistical processing like that carried out in optical analysis technologies such as FCS, FIDA or PCH.
  • an object of the present invention is to provide a method for detecting fluorescent particles with higher sensitivity by using a novel optical analysis technology that allows detection of the status or properties of target particles in a sample solution in which the concentration or number density of the target particles is at a lower level than the level used in the aforementioned optical analysis technologies.
  • the inventors of the present invention found that, detecting a fluorescent particle dispersed and moving randomly in a sample solution, by detecting the fluorescent particle using a scanning molecule counting method, a fluorescent particle can be detected with favorable sensitivity even in the case the concentration of the target particle in the sample solution is extremely low, and further that a fluorescent particle can be detected with even higher sensitivity by carrying out measurement according to the scanning molecule counting method in the presence of a triplet excited state quenching agent.
  • the present invention provides:
  • the method for detecting a fluorescent particle wherein in the step for individually detecting target particles by detecting a light signal from the individual fluorescent particles, the entry of a single fluorescent particle into the photodetection region is detected based on the form of a detected chronological light signal.
  • the scanning molecule counting method used in the method for detecting a fluorescent particle of an embodiment of the present invention statistical processing involving the calculation of fluctuations in fluorescence intensity is not carried out. Consequently, according to the method for detecting a fluorescent particle of the present invention, an analysis target in the form of a fluorescent particle in a sample can be detected even in cases in which the fluorescent particle is only present in an extremely small amount in the sample. Moreover, in the method for detecting a fluorescent particle of the present invention, a fluorescent particle can be detected with extremely high sensitivity by carrying out measurement according to a scanning molecule counting method in the presence of a triplet excited state quenching agent.
  • the scanning molecule counting method consists of detecting light emitted from particles that emit light dispersed and moving randomly in a sample solution (to be referred to as "luminescent particles") present in a microregion when the luminescent particles cross through the microregion.
  • the scanning molecule counting method makes it possible to acquire information relating counting of luminescent particles or the concentration or number density of luminescent particles in a sample solution by individually detecting each luminescent particle in the sample solution. It goes without saying that only an extremely small amount (for example, on the order of only several tens of microliters) of sample is required for measurement in the same manner as optical analysis technologies such as FIDA, and measurement time is short.
  • properties such as concentration or number density can be quantitatively detected for luminescent particles at a lower concentration or number density in comparison with the case of optical analysis technologies such as FIDA.
  • luminescent particles refer to particles that emit light by fluorescence, phosphorescence, chemiluminescence, bioluminescence or light scattering and the like.
  • luminescent particles in the form of fluorescent particles are targeted for detection.
  • a "photodetection region" of a confocal microscope and multi-photon microscope refers to a microregion in which light is detected by a confocal microscope of multi-photon microscope, and in the case illumination light is reflected from an object lens, the region where that illumination light is focused corresponds to a microregion. Furthermore, this microregion is defined by the positional relationship between the object lens and pinhole in a confocal microscope in particular.
  • Light is successively detected while moving the location of the photodetection region in a sample solution, or in other words, while scanning the sample solution by photodetection regions.
  • the photodetection region being moved contains a luminescent probe bound to or associated with a randomly moving particle
  • light from the luminescent probe is detected.
  • the luminescent probe may also dissociate from the particle to be detected during detection of light after having bound to that particle. Light signals from the luminescent probe are individually detected in the successively detected light.
  • the presence of an individual particle or a particle bound to the luminescent probe is successively detected and various information relating to the state of the particle in the solution is acquired. More specifically, in the aforementioned configuration, the number of particles detected during movement of the location of the photodetection region may also be counted, for example, by counting individually detected particles (particle counting). According to this configuration, information relating to number density or concentration of particles in a sample solution is obtained by combining the number of particles and the amount of movement of the location of the photodetection region.
  • particle number density or concentration can be specifically calculated by, for example, moving the location of the photodetection region at a prescribed speed by an arbitrary method, and specifying the total volume of the movement locus of the location of the photodetection region.
  • a ratio of relative number density or concentration may also be calculated relative to a plurality of sample solutions or reference sample solution having a standard concentration or number density.
  • the scanning molecule counting method by employing a configuration that allows the location of the photodetection region to be moved by changing the light path of the optical system, movement of the photodetection region is rapid, and mechanical vibrations or actions attributable to fluid dynamics do not substantially occur in the sample solution.
  • a judgment as to whether or not a luminescent probe bound to a single particle has entered the photodetection region based on successively detected light signals may be carried out based on the form of a detected chronological light signal.
  • the entry of a luminescent probe bound to a single particle into the photodetection region may be typically detected when a light signal has been detected that has intensity greater than a prescribed threshold value.
  • a luminescent probe bound to a single particle includes the state in which a single luminescent probe is bound to a single particle, a state in which a plurality of luminescent probes are bound to a single particle, and a state in which a luminescent probe has dissociated from a particle after having bound to a single particle depending on the mode of the experiment.
  • the movement speed of the location of the photodetection region in a sample solution may be suitably changed based on the properties of the luminescent probe bound to a particle or the number density or concentration thereof in a sample solution.
  • the mode of light detected from a luminescent probe bound to a particle can be changed according to the properties thereof or the number density or concentration in a sample solution.
  • the movement speed of the photodetection region is preferably suitably changed so that light from the luminescent probe bound to a single particle can be measured with favorable accuracy and sensitivity.
  • the movement speed of the location of the photodetection region in a sample solution is preferably set to be faster than the diffusion movement speed (average speed of particles moving by Brownian movement) of the luminescent probe bound to a particle to be detected.
  • the diffusion movement speed average speed of particles moving by Brownian movement
  • the luminescent probe bound to a particle moves randomly through a solution by Brownian movement and enters and leaves the photodetection region a plurality of times, light signals (light signals indicating the presence of a particle desired to be detected) end up being detected a plurality of times from a single luminescent probe, thereby making it difficult to make a detected light signal correspond to the presence of a single particle desired to be detected. Therefore, as was previously described, the movement speed of the photodetection region is set to be faster than the diffusion movement speed of the luminescent probe bound to a particle, and as a result thereof, a luminescent probe bound to a single particle can be made to correspond to a single light signal (light signal representing the presence of a particle).
  • the movement speed of the photodetection region is set so as to a speed faster than the diffusion movement speed of a detection target in the form of a fluorescent particle. Furthermore, since diffusion movement speed varies according to the luminescent probe bound to a particle, the movement speed of the photodetection region is preferably suitably changed corresponding to the properties (and particularly, the diffusion constant) of the luminescent probe bound to a particle as previously described.
  • Changing of the light path of the optical system to move the location of the photodetection region may be carried out by an arbitrary method.
  • the location of the photodetection region may be changed by changing the light path using a galvanometer mirror employed in laser scanning optical microscopes.
  • the movement locus of the location of the photodetection region may be set arbitrarily, and for example, can be selected from among, for example, a circular, oval, rectangular, linear or curved locus.
  • the photodetection mechanism per se is composed so as to detect light from a photodetection region of a confocal microscope or multi-photon microscope in the same manner as in the case of optical analysis technologies such as FIDA, the amount of sample solution may also be similarly an extremely small amount.
  • optical analysis technology employing the scanning molecule counting method can be applied to sample solutions in which the number density or concentration of particles is considerably lower than that required by optical analysis technologies such as FIDA.
  • each particle dispersed or dissolved in a solution is detected individually. Consequently, counting of particles, determination of particle concentration or number density in a sample solution, or acquisition of information relating to concentration or number density, can be carried out quantitatively using that information. Namely, according to the scanning molecule counting method, since particles are detected one at a time by creating a 1:1 correlation between a particle passing through a photodetection region and a detected light signal, particles dispersed and moving randomly in a solution can be counted. Thus, according to the scanning molecule counting method, the concentration or number density of particles in a sample solution can be determined more accurately than in the prior art.
  • the fluorescent particles can be detected even if the concentration of fluorescent particles in a sample solution is lower than the concentration able to be determined based on fluorescence intensity as measured with a fluorescence spectrophotometer or plate reader.
  • the inside of the sample solution is observed uniformly or the sample solution is observed in a mechanically stable state without imparting mechanical vibrations or actions attributable to fluid dynamics to the sample solution. Consequently, the reliability of quantitative detection results is improved in comparison with the case of causing the generation of flow in a sample, and particles to be detected in a sample solution can be measured in a state that is free of effects caused by dynamic action and artifacts.
  • the configuration of the device becomes complex. In addition, the amount of sample required increases considerably.
  • the particles in solution, luminescent probe, complex thereof or other substances may undergo deterioration or degeneration due to the fluid dynamic action generated by that flow.
  • the basic configuration of the scanning molecule counting method can be realized by an optical analysis device composed by combining the optical system of a confocal microscope capable of performing FCS or FIDA and the like with a photodetector.
  • an optical analysis device 1 is composed of optical system components 2 to 17, and a computer 18 for controlling the operation of each component of the optical systems and acquiring and analyzing data.
  • the optical system of the optical analysis device 1 may be composed in the same manner as the optical system of an ordinary confocal microscope, wherein laser light (Ex) that has propagated from a light source 2 through a single-mode optic fiber 3 is radiated in the form of light that is emitted at an angle determined according to a characteristic numerical aperture (NA) at the outgoing end of the fiber, the laser light is converted to parallel light by a collimator 4 and is reflected by a dichroic mirror 5 and reflecting mirrors 6 and 7, after which it enters an object lens 8.
  • a microplate 9, in which are arranged sample containers or wells 10 into which are dispensed one to several tens of microliters of a sample solution, is typically arranged above the object lens 8.
  • Laser light emitted from the object lens 8 is focused on the sample solution in the sample containers or wells 10, forming a region of high light intensity (excitation region).
  • Target particles a luminescent probe that binds to the particles, and typically a molecule having a luminescent label such as a fluorescent dye added thereto, are dispersed or dissolved in the sample solution.
  • the luminescent probe When a particle bound to or associated with the luminescent probe (or the luminescent probe may dissociate from the particle after having initially bound thereto depending on the mode of the experiment) enters the excitation region, the luminescent probe is excited and light is released during that time.
  • the released light (Em) passes through the object lens 8 and dichroic mirror 5, is reflected by a mirror 11, is concentrated by a condenser lens 12 and then passes through a pinhole 13 followed by passing through a barrier filter 14 (here, only light components of a specific wavelength band are selected), after which the released light is introduced into a multi-mode optic fiber 15 and reaches a photodetector 16, and after being converted to a chronological electrical signal, is input to the computer 18 followed by undergoing processing for optical analysis by a mode to be subsequently explained.
  • the pinhole 13 is arranged at a location conjugate to the focal position of the object lens 8 as is known by persons skilled in the art.
  • the focused region of the laser light exemplified in FIG. 1B is normally a photodetection region in the present optical analysis device having an effective volume of about 1 fL to 10 fL, and is referred to as the confocal volume (and typically has a Gaussian distribution or Lorentzian distribution in which light intensity reaches a peak in the center of the region, and effective volume is the volume of a roughly ellipsoidal shape in which the boundary of light intensity is a plane defined as 1/e2).
  • an ultra-high-sensitivity photodetector capable of use in photon counting is preferably used for the photodetector 16.
  • the stage of the microscope (not shown) may be provided with a stage position adjustment device 17a for moving the position of the microplate 9 in the horizontal direction in order to change the well 10 to be observed. Operation of the stage position adjustment device 17a may be controlled by the computer 18. As a result of employing this configuration, measurements can be carried out rapidly even in the case of multiple specimens.
  • a mechanism is provided for scanning the sample solution by photodetection regions by changing the light path of the optical system, namely a mechanism is provided for moving the location of the focused region (photodetection region) in the sample solution.
  • a mirror light deflector 17 that changes the orientation of the reflecting mirror 7, for example, may be employed as a mechanism for moving the location of the photodetection region in this manner as schematically exemplified in FIG. 1C .
  • This mirror light deflector 17 may be composed in the same manner as a galvanometer mirror device provided in ordinary laser scanning optical microscopes.
  • the mirror light defector 17 is driven in coordination with light detection by the photodetector 16 under the control of the computer 18 so as to achieve a desired movement pattern of the location of the photodetection region.
  • the movement locus of the location of the photodetection region may be arbitrarily selected from among a circular, oval, rectangular, linear and curved locus or a combination thereof (and various movement patterns may be allowed to be selected by a program in the computer 18).
  • the location of the photodetection region may be moved in the vertical direction by moving the object lens 8 up and down.
  • the aforementioned optical system is used in the form of a multi-photon microscope. In that case, since light is only released in the focused region of the excitation light (photodetection region), the pinhole 13 may be omitted.
  • optical system components 2 to 5 for generating excitation light may be omitted.
  • the aforementioned optical system of a confocal microscope is used as is.
  • a plurality of excitation light sources 2 are provided as shown in FIG. 1A , and these may be composed so as allow the wavelength of the excitation light to be suitably selected according to the wavelength of light that excites a conjugate of a particle and luminescent probe or a luminescent probe.
  • the light emitted therefrom may be detected separately according to wavelength.
  • spectral analysis technologies such as FIDA are superior in that they require only an extremely small amount of sample and allow testing to be carried out rapidly.
  • the concentration and properties of target particles are in principle determined based on fluctuations in fluorescence intensity.
  • the concentration or number density of target particles in a sample solution is required to be of a level such that roughly one target particle is present at all times in a photodetection region CV during measurement of fluorescence intensity, and that significant light intensity (photon count) be detected at all times during the measurement time.
  • the concentration or number density of the target particles is lower than that level, such as in the case of being at a level such that a target particle only occasionally enters the photodetection region CV, significant light intensity (photon count) only appears during a portion of the measurement time, thereby making it difficult to accurately determine fluctuations in light intensity.
  • the concentration of target particles is considerably lower than the level at which roughly one target particle is present in the photodetection region at all times during measurement, determination of fluctuations in light intensity are subject to background effects, thereby prolonging measurement time in order to obtain an adequate amount of significant light intensity data for making a determination.
  • the concentration, number density or other properties of target particles can be detected even in the case the concentration of target particles is lower than the level required by spectral analysis technologies such as FIDA.
  • photodetection is carried out by changing the light path by driving a mechanism (mirror light defector 17) for moving the location of the photodetection region while moving the location of the photodetection region CV in a sample solution, or in other words, while scanning the interior of a sample solution by photodetection regions CV, as is schematically depicted in FIG. 2A .
  • a mechanism mirror light defector 17
  • FIG. 2A for example, when the optical analysis device passes a region in which a single particle (a luminescent probe in the form of a fluorescent dye is bound to the particle in the drawing) is present (tl) during the time the photodetection region CV moves (time t0 to t2 in the drawing), significant light intensity (Em) is detected as depicted in FIG. 2B .
  • a single particle a luminescent probe in the form of a fluorescent dye is bound to the particle in the drawing
  • FIG. 2B significant light intensity
  • particles bound with a luminescent probe are individually detected, and by counting the number of those particles, the number of particles present in a measured region or information relating to concentration or number density can be acquired.
  • individual particles are detected without carrying out statistical arithmetic processing so as to calculate fluctuations in fluorescence intensity.
  • information relating to particle concentration or number density can be acquired even in a sample solution in which the concentration of particles to be observed is so low that they cannot be analyzed by FIDA and the like with adequate accuracy.
  • measurements can be carried out at a lower concentration than in the case of measuring the concentration of fluorescent-labeled particles based on fluorescence intensity measured with a fluorescence spectrophotometer or plate reader.
  • fluorescence intensity is normally assumed to be proportional to the concentration of the fluorescent-labeled particles.
  • particle concentration measurement accuracy improves for high particle concentrations to a greater degree than conventional methods consisting of determining concentration based on the assumption of fluorescence intensity being proportional to the concentration of fluorescent-labeled particles.
  • a plurality of luminescent probes are bound to a single target particle, when a certain amount of luminescent probe is added to the sample solution, the number of luminescent probes that bind to the particles undergoes a relative decrease as the concentration of target particles increases.
  • Measurement of light intensity in optical analyses using the scanning molecule counting method may also be carried out by a mode similar to the fluorescence intensity measurement step of FCS or FIDA with the exception of moving the location of a photodetection region in a sample solution (scanning the interior of the sample solution) by driving the mirror light deflector 17 during measurement.
  • sample solution is typically injected into the wells 10 of the microplate 9, and after placing the microplate 9 on the microscope stage, a user inputs instructions for starting measurement to the computer 18.
  • the computer 18 initiates radiation of excitation light and measurement of light intensity in a photodetection region in the sample solution in accordance with a program (consisting of a procedure for changing the light path so as to move the location of the photodetection region in the sample solution and a procedure for detecting light from the photodetection region during movement of the location of the photodetection region) stored in a memory device (not shown).
  • a program consisting of a procedure for changing the light path so as to move the location of the photodetection region in the sample solution and a procedure for detecting light from the photodetection region during movement of the location of the photodetection region
  • the mirror light deflector 17 drives the mirror 7 (galvanometer mirror) under the control of a processing operation in accordance with the program of the computer 18, and the location of the photodetection region is moved in the wells 10.
  • the photodetector 16 converts successively detected light to electrical signals and transmits those signals to the computer 18.
  • chronological light intensity data is generated from the transmitted light signals and stored therein.
  • the photodetector 16 is typically an ultra-high-sensitivity photodetector capable of detecting the arrival of a single photon. Accordingly, light detection is in the form of photon counting that is carried out in a mode in which the number of photons arriving at the photodetector in a prescribed unit time period (bin time), such as every 10 ⁇ s, is successively measured over a prescribed amount of time, and chronological light intensity data is in the form of chronological photon count data.
  • the movement speed when moving the location of the photodetection region during measurement of light intensity may be an arbitrary speed, and for example, may be a prescribed speed set experimentally or so as to comply with the analysis objective.
  • the region through which the photodetection region passes is required to have a certain size or volume. Consequently, the location of the photodetection region is moved by a mode that allows movement distance to be determined.
  • the presence of a proportional relationship between elapsed time during measurement and movement distance of the location of the photodetection region facilitates interpretation of measurement results. Accordingly, although movement speed is basically made to preferably be a constant speed, it is not limited thereto.
  • the movement speed of the location of the photodetection region is preferably set to value that is faster than the random movement speed of the target particles (and more precisely, conjugates of particles and luminescent probe or luminescent probe that has degraded and been released after binding with the particles, and in the present invention, fluorescent particles), or in other words, a speed faster than movement speed attributable to Brownian movement.
  • target particles in an optical analysis technology using the scanning molecule counting method are particles that are dispersed or dissolved in a solution and randomly move about freely therein, their locations move over time due to Brownian movement.
  • the movement speed of the location of the photodetection region is preferably set to be faster than the average movement speed attributable to Brownian movement (diffusion movement speed) so that particles cross the photodetection region in nearly a straight line as depicted in FIG. 4A .
  • a profile of the change in light intensity corresponding to individual particles becomes nearly uniform as exemplified in FIG. 4B in the chronological light intensity data, and a correlation between individual target particles and light intensity can be easily determined.
  • the profile of changes in light intensity is roughly the same as the distribution of excitation light intensity.
  • the diffusion coefficient D of a target particle is predicted to be about 2.0 ⁇ 10 -10 m 2 /s, if Wo is about 0.62 ⁇ m, then Vdif becomes 1.0 ⁇ 10 -3 m/s. Consequently, the movement speed of the location of the photodetection region is set to a value of 15 mm/s, which is about 10 times greater than that.
  • a preferable movement speed of the location of the photodetection region is determined by repeatedly carrying out preliminary experiments in order to find those conditions under which the prolife of changes in light intensity become the predicted profile (and typically, a prolife that is roughly the same as the excitation light distribution) by trying various settings for the movement speed of the location of the photodetection region.
  • the computer 18 analyzes light intensity in the manner described below by carrying out processing in accordance with a program stored in a memory device (consisting of a procedure for individually detecting light signals corresponding to individual luminescent particles from detected light).
  • a threshold value Io is set for light intensity, and when a duration ⁇ during which light intensity continuously exceeds that threshold value is within a prescribed range, that profile of light intensity is judged to correspond to the passage of a single particle through the photodetection region, and that single target particle is detected.
  • the threshold value Io for light intensity and the prescribed range of duration ⁇ are determined based on a profile presumed to be the intensity of light emitted from a conjugate of an target particle and luminescent probe (or a luminescent probe that has been degraded and separated after binding with that particle) that moves at a prescribed speed relative to the photodetection region. Those specific values may be arbitrarily set experimentally, or may be selectively determined according to the properties of the conjugate of the target particle and luminescent probe (or a luminescent probe that has been degraded and separated from the particle).
  • Counting of target particles is carried out by counting the number of particles detected according to the aforementioned techniques for detecting target particles by an arbitrary method. However, in the case of a large number of particles, counting may be carried out according to processing exemplified in FIGS. 5 and 6B .
  • one example of a method for counting particles from chronological light intensity (photon count) data consists of measuring light intensity as explained above. Namely, chronological light signal data (photon count data) is acquired by carrying out scanning of a sample solution by photodetection regions and counting the number of photons (Step 100) . Smoothing processing (Step 110, "Smoothing" in the second graph from the top in FIG. 6B ) is then carried out on the chronological light signal data ("Detection result (unprocessed)" in the top graph of FIG. 6B ).
  • Smoothing processing is carried out by, for example, the moving average method. Furthermore, parameters used when carrying out smoothing processing, such as the number of data points averaged at one time or the number of times movement is averaged in the case of the moving average method, are suitably set corresponding to the movement speed of the location of the photodetection region when acquiring light intensity data (scanning speed) and bin time.
  • a first derivative is calculated for the time of the chronological light signal data following smoothing processing (Step 120).
  • the change in the time derivative of chronological light signal data increases at the inflection point of the signal value as exemplified by "Time differentiation" in the second graph from the bottom in FIG. 6B . Consequently, the starting point and ending point of a significant signal (peak signal) can be advantageously determined by referring to this time derivative.
  • a peak region is identified by seeking and determining the starting point and ending point of a single peak signal by successively referring to time derivatives in the chronological time-differentiated data of the chronological light signal data (Step 130) .
  • a bell-shaped function is fit to the smoothened chronological light signal data in that peak region (Bell-shaped function fitting" in the bottom graph of FIG. 6B ) .
  • parameters such as peak intensity Imax of the bell-shaped function, peak width (full width at half maximum) w and correlation coefficient (of the least squares method) during fitting are calculated (Step 140) .
  • the bell-shaped function subjected to fitting is typically a Gaussian function, it may also be a Lorentzian function.
  • a judgment is then made as to whether or not the calculated bell-shaped function parameters are within a presumed range for the parameters of a bell-shaped profile depicted by a light signal detected when a single conjugate of a particle and luminescent probe or luminescent probe has passed through a photodetection region, namely whether or not peak intensity, peak width and correlation coefficient are each within a prescribed range (Step 150).
  • the searching and discrimination of peak signals in the aforementioned processing of steps 130 to 160 are carried out repeatedly for the entire range of chronological light signal data, and each time a single target particle is detected, that a target particle is counted as a particle.
  • the particle count value obtained up to that time is taken to be the number of target particles detected in the chronological light signal data.
  • the number density or concentration of the target particles is determined using the total volume of the photodetection region traversed by the target particles during acquisition of chronological light signal data.
  • the effective volume of the photodetection region fluctuates dependent upon the wavelength of the excitation light or detection light, numerical aperture of the lens, and adjusted state of the optical system, it is generally difficult to determine the number density or concentration of target particles from design values. Thus, it is not easy to determine the total volume of the traversed region of a photodetection region.
  • a plurality of solutions having different concentrations may be provided for use as reference solutions, measurements may be carried out on each reference solution, and the average value of the calculated Vt of each may be used as the total volume Vt of the traversed region of the photodetection region.
  • the optical analysis device of the present invention may preliminarily store information on the relationship between concentration C and particle count N (Equation (5)) for various standard particles and for presumed photodetection region movement patterns in a memory device of the computer 18, and may be configured so that a device user is able to use that suitably stored relationship information when performing optical analyses.
  • the method for detecting a fluorescent particle of the present invention is a method for detecting a fluorescent particle dispersed and randomly moving in a sample solution, wherein the fluorescent particle is detected by counting according to the scanning molecule counting method in the presence of a substance that promotes transition of the fluorescent particles from a triplet excited state to a singlet ground state. Since the scanning molecule counting method is a measurement method that enables luminescent particles to be measured one particle at a time while molecules are in a discrete state, measurements can be carried out on luminescent particles at a comparatively low concentration on the pM order or lower.
  • the method for detecting fluorescent particles of the present invention can be used to count fluorescent particles with high sensitivity.
  • the method for detecting a fluorescent particle of the present invention is capable of detecting a fluorescent particle with extremely high sensitivity by measuring according to the scanning molecule counting method in the presence of a substance that promotes transition from a triplet excited state to a singlet ground state (triplet excited state quenching agent).
  • the molecules in a ground state When molecules in a ground state are irradiated with light, the molecules enter a singlet excited state after which they again return to the ground state.
  • the first is a path by which molecules return directly to the ground state from the singlet excited state, and the light released at this time is fluorescent light.
  • the other path is a path by which molecules return to the ground state from the singlet excited state by going through a triplet excited state, and fluorescence is not emitted from molecules that return to the ground state by the aforementioned path.
  • the lifetime of the fluorescent light is known to be on the nanosecond order for most molecules.
  • the duration of the path by which molecules return to the ground state from the singlet excited state by going through a triplet excited state (without emitting fluorescent light) is generally on the microsecond order.
  • waste results due to a portion of the molecules excited to the singlet excited state returning to the ground state by going through the triplet excited state.
  • a triplet excited state quenching agent improves fluorescent brightness by utilizing the fact that it collides more frequently with molecules in the triplet excited state than molecules in the singlet excited state due to differences in length between lifetime of the singlet excited state and lifetime of the triplet excited state. More specifically, as a result of measuring according to the scanning molecule counting method in the presence of a triplet excited state quenching agent, fluorescent particles in the triplet excited state present in a sample solution are returned to the ground state by the triplet excited state quenching agent. Fluorescent particles returned to the ground state are again excited to the singlet excited state when irradiated with light. Namely, as a result of rapidly eliminating the triplet excited state that does result in emission of fluorescent light, fluorescent brightness can be improved, thereby making it possible to detect fluorescent particles with higher sensitivity.
  • triplet excited state quenching agent used in the present invention is a substance that has an action that returns molecules in the triplet excited state to the ground state.
  • the triplet excited state quenching agent is used by suitably selecting from among known triplet excited state quenching agents in consideration of such factors as the type of fluorescent particles, wavelength of the excitation light, and wavelength of the fluorescent light.
  • triplet excited state quenching agents include potassium iodide (KI), cysteamine (also known as 2-aminoethanethiol) and cyclooctatetraene.
  • potassium iodide and cysteamine are used preferably.
  • one type of triplet excited state quenching agent may be used alone or two or more types may be used in combination.
  • the fluorescence detection wavelengths include at least a portion of the wavelength range of 510 nm to 560 nm
  • one or more types of triplet excited state quenching agents selected from among potassium iodide and cysteamine are preferably used for the triplet excited state quenching agent.
  • cysteamine is preferably used for the triplet excited state quenching agent.
  • fluorescent particles for which the fluorescence wavelength includes at a least a portion of the wavelength range of 510 nm to 560 nm include fluorescent substances such as Rhodamine GreenTM, Alexa FluorTM 488, fluorescein, FITC, 5-carboxyfluorescein (5-FAM) or Cy2TM, and particles in which these fluorescent substances are bound thereto.
  • fluorescent particles for which the fluorescence wavelength includes at least a portion of the wavelength range of 560 nm to 620 nm include fluorescent substances such as TAMRATM, Rhodamine, Cy3TM, Alexa FluorTM 546 or Alexa FluorTM 555, and particles in which these fluorescent substances are bound thereto.
  • a method for quantitative determination of a target particle of the present invention includes the following steps (a) and (b):
  • a particle dispersed and moving randomly in a sample solution refer to a particle such as an atom, a molecule or an aggregates thereof dispersed or dissolved in a sample solution (and may be a particle that emits light or a particle that does not emit light) that moves about freely by Brownian movement in a solution without being immobilized on a substrate and the like.
  • the fluorescent particles targeted for detection with the method for detecting a fluorescent particle of the present invention are a substance that releases fluorescent light as a result of being irradiated with light of a specific wavelength, and examples thereof include fluorescent substances such as fluorescent dyes or quantum dots.
  • the method for detecting a fluorescent particle of the present invention is also able to detect a non-fluorescent substance by using a fluorescent probe. More specifically, a fluorescent probe is used that has a site that specifically or non-specifically binds or adsorbs to a detection target in the form of a target particle (non-fluorescent substance) and emits fluorescence when bound to the target particle.
  • the fluorescent probe and target particle are bound by adding the fluorescent probe to a sample solution containing the target particle.
  • the formed fluorescent probe bound with the target particle is a fluorescent particle, and can be detected by the method for detecting a fluorescent particle of the present invention.
  • Target particles refer to particles that are dispersed and moving randomly in a sample solution, and examples thereof include biomolecules such as proteins, peptides, nucleic acids, nucleic acid-like substances, lipids, saccharides, amino acids or aggregates thereof, particulate biological targets such as viruses or bacteria, and non-biological particles (such as atoms, molecules, micelles or metal colloids).
  • Nucleic acids may be DNA or RNA, or may be artificially amplified substances in the manner of cDNA.
  • nucleic acid-like substances examples include substances in which side chains and the like of naturally-occurring nucleotides in the manner of DNA or RNA (nucleotides present in nature) have been modified by functional groups such as an amino group, and substances that have been labeled with a protein or low molecular weight compound and the like.
  • nucleic acid-like substances include bridged nucleic acids (BNA), nucleotides in which an oxygen atom at position 4' of a naturally-occurring nucleotide has been substituted with a sulfur atom, nucleotides in which a hydroxyl group at position 2' of a naturally-occurring nucleotide has been substituted with a methoxy group, hexitol nucleic acids (HNA) and peptide nucleic acids (PNA).
  • BNA bridged nucleic acids
  • HNA hexitol nucleic acids
  • PNA peptide nucleic acids
  • a fluorescent probe that binds with a target particle is preferably a substance in which luminescence properties of the released light differ between the state in which it is bound to a target particle and the state in which it is present alone.
  • a fluorescent probe having different luminescence properties between the state in which it is bound to a target particle and the state in which it is present alone means that the intensity of light of specific wavelength differs between the state in which it is bound to a target particle and the state in which it is present alone. Differing the intensity of a light of a specific wavelength between the state in which the fluorescent probe is bound to a target particle and the state in which it is present alone (such as differing in fluorescence intensity) makes it possible to distinguish between the two during detection according to the scanning molecule counting method.
  • examples of the fluorescent probe include that in which a fluorescent substance is bound to an oligonucleotide that hybridizes with the target particle, a nucleic acid-binding protein bound with a fluorescent substance, and a fluorescent dye molecule that binds to nucleic acid.
  • the aforementioned oligonucleotide may be DNA, RNA or an artificially amplified substance in the manner of cDNA, or a substance that contains a portion or all of a nucleic acid-like substance capable of forming a nucleotide chain and base pairs in the same manner as naturally-occurring nucleic acid bases.
  • the target particle is a protein
  • a substance obtained by labeling an antigen or antibody to the target particle or a ligand or receptor of the target particle with a fluorescent substance can be used as a fluorescent probe.
  • binding of a fluorescent substance to a substance that specifically or non-specifically binds or absorbs to a target particle such as a nucleic acid or protein can be carried out by ordinary methods.
  • the fluorescent probe that binds to a target particle may be that which non-specifically binds to a target particle, from the viewpoint of accuracy of detection and quantitative determination of target particles, a fluorescent probe that binds specifically is preferable. Furthermore, the fluorescent probe that specifically binds to a target particle is only required to be that which preferentially binds to the target particle rather than binding to other substances having physical or chemical properties similar to those of the target particle, and is not required to be that which does not bind at all to substances other than the target particle.
  • an oligonucleotide labeled with a fluorescent substance used as a fluorescent probe may have a base sequence that is completely complementary to the base sequence of the target particle, or may have a base sequence that contains mismatches with the base sequence of the target particle.
  • the target particle is a protein
  • a dye such as a fluorescent dye in the manner of hydrophobic probes ANS, MANS and TNS
  • the fluorescent probe per se is not necessarily required to emit fluorescent light.
  • the target particle is a nucleic acid or nucleic acid-like substance
  • an oligonucleotide that hybridizes with the target particle as a fluorescent probe
  • luminescence properties can be made to differ between the state in which the fluorescent probe is present alone and the state in which the fluorescent probe is bound to the target particle.
  • fluorescent double-stranded nucleic acid-like substances that specifically bind to a double-stranded structure include fluorescent intercalators and groove binders bound to a fluorescent substance.
  • substances composed of at least two constituents that emit fluorescence due a mutual positional change in at least two of the aforementioned constituents as a result of binding to a target particle may also be employed as fluorescent probes.
  • fluorescent probes include fluorescent proteins that undergo a structural change and release strong fluorescence when binding to a certain particle, and molecules that aggregate and form a fluorescent metal complex when binding to a certain molecule (complex ligands).
  • complex ligands complex ligands
  • luminescence properties can also be made to differ between a fluorescent probe present alone in a sample solution and a fluorescent probe in a state of being bound to a target particle by using fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • a fluorescent substance serving as an energy donor in FRET and a fluorescent substance serving as an energy acceptor can be used as substances that bind to a target particle, and a substance for which FRET occurs in a state in which a fluorescent probe is present alone but for which FRET is not allowed to occur in the state of being bound to the target particle can be used as a fluorescent probe. Since FRET does not occur in the fluorescent probe bound to the target particle, fluorescence is released from the fluorescent substance serving as the energy donor.
  • fluorescence released from the fluorescent substance serving as the energy donor is either not detected from the fluorescent probe present alone or that fluorescence is weak. Therefore, by detecting fluorescence released from the fluorescent substance serving as the energy donor, a target particle bound to the fluorescent probe can be distinguished from the fluorescent probe present alone and thereby detected.
  • a fluorescent substance serving as an energy donor in FRET and a substance serving as an energy acceptor can be preferably used as oligonucleotides that form an intramolecular structure when in the state of a single-stranded nucleic acid molecule, and a molecular beacon probe bound such that FRET occurs when in the state of a single-stranded nucleic acid molecule, but does not occur when in the state of an association product formed by hybridizing with another single-stranded nucleic acid molecule, can be preferably used as a fluorescent probe.
  • a substance is preferably used that has a fluorescent substance serving as an energy donor or a substance serving as an energy acceptor bound to the 3'-terminal side with the remaining other of the pair bound to the 5'-terminal side, has base sequences that are mutually complementary to the region of the 3'-terminal side and 5'-terminal side, and forms an intramolecular structure (a so-called stem-loop structure) by forming base pairs in these base sequences.
  • the mutually complementary regions that form the intramolecular base pairs of the molecular beacon probe are present so as to interpose a region that hybridizes with a target particle, and the region on the 3'-terminal side and the region on the 5'-terminal side may be regions that respectively contain the 3'-terminal or 5'-terminal or regions that do not.
  • the number of bases and base sequence of the regions that form the base pairs are to such a degree that the stability of the formed base pairs is lower than the stability of the association product with the target particle and base pairs can be formed under the measurement conditions.
  • a fluorescent probe present alone can also be distinguished from a fluorescent probe bound to a target particle by using a fluorescent double-stranded nucleic acid-binding substance that specifically binds to a double-stranded structure and inducing FRET between the fluorescent double-stranded nucleic acid-binding substance and a fluorescent substance labeled with the fluorescent probe.
  • a fluorescent double-stranded nucleic acid-binding substance or a fluorescent substance labeled with the fluorescent probe serves as a FRET energy donor while the other serves as a FRET energy acceptor. Fluorescence released from the fluorescent substance used to label the fluorescent probe is detected from the fluorescent probe present alone.
  • the fluorescent double-stranded nucleic acid-binding substance binds to the fluorescent probe bound to a target particle, fluorescent released by FRET is detected from the conjugate thereof, and as a result thereof, the conjugate can be distinguished from the fluorescent probe present alone, thereby enabling its detection.
  • the fluorescent probe is preferably designed so that the region that forms a double-strand in the association product of the fluorescent probe and target particle is 400 bp or less.
  • two types of fluorescent probes may also be used in the present invention.
  • the target particle is a nucleic acid or nucleic acid-like substance
  • two types of fluorescent probes are designed so as to hybridize mutually adjacent to a target particle, one of the fluorescent probes is labeled with a fluorescent substance serving as an energy donor in FRET, while the other fluorescent probe is labeled with a substance serving as an energy acceptor in FRET.
  • FRET does not occur in the case the fluorescent probes are present alone, the two types of fluorescent probes are mutually brought into close proximity thereby resulting in the occurrence of FRET as a result of binding to the target particle. Consequently, the target particle bound to the fluorescent probe can be detected by detecting fluorescence released by FRET.
  • a sample solution is prepared that contains fluorescent particles and a fluorescent probe that binds to a triplet excited state quenching agent that acts on the fluorescent particles. More specifically, the sample solution is prepared by adding the fluorescent particles and triplet excited state quenching agent to a suitable solvent.
  • a suitable solvent There are no particular limitations on the solvent provided it does not impair detection of light released from the fluorescent particles or detection of the fluorescent probe according to the scanning molecule counting method, and can be used by suitably selecting from among buffers commonly used in the aforementioned technical field. Examples of these buffers include phosphate buffers such as phosphate-buffered saline (PBS, pH 7.4) and Tris buffers.
  • the triplet excited state quenching agent is added to the sample solution at a concentration sufficient for demonstrating action that returns molecules from the triplet excited state to the ground state.
  • concentration at which quenching action is demonstrated can be determined experimentally. More specifically, the triplet excited state quenching agent is added at various concentrations to a measurement solution containing a prescribed amount of fluorescent particles (which may be particles containing the same type of fluorescent substance as the fluorescent particles targeted for detection) followed by measuring the percentage of molecules in the triplet excited state in the fluorescent probes of each measurement solution.
  • the concentration at which the percentage of molecules in the triplet excited state is significantly lower than that of a measurement solution to which the triplet excited state quenching agent has not been added is the concentration at which quenching action is demonstrated by the aforementioned triplet excited state quenching agent.
  • the percentage of molecules in the triplet excited state can be determined by FCS measurement.
  • the concentration of the triplet excited state quenching agent during measurement by the scanning molecule counting method is preferably 0.1 mM to 20 mM, more preferably 0.3 mM to 10 mM, and even more preferably 0 . 5 mM to 5 mM.
  • the combined concentration of both is preferably within the aforementioned ranges.
  • a sample solution may be prepared by adding a triplet excited state quenching agent to a solution containing fluorescent particles obtained by preliminarily binding target particles and a fluorescent probe.
  • fluorescent particles may also be formed by preparing a sample solution obtained by adding target particles, fluorescent probe and triplet excited state quenching agent, followed by binding the target particles and fluorescent probe in the aforementioned sample solution.
  • the target particles and fluorescent probe can be bound in the aforementioned sample solution simply by incubating the aforementioned sample solution for a prescribed amount of time as necessary.
  • the target particles and fluorescent probe are nucleic acids or nucleic acid-like substances having a double-stranded structure
  • the target particles and luminescent probe are preferably associated after having denatured the nucleic acid and the like in the sample solution.
  • “denaturing a nucleic acid molecule or nucleic acid-like substance” refers to dissociation of base pairs. For example, this refers to dissociating base pairs formed by mutually complementary base sequences in a molecular beacon probe to disassemble an intramolecular structure and form a single-stranded structure, or converting a double-stranded nucleic acid molecule into a single-stranded nucleic acid molecule.
  • the fluorescent probe is an oligonucleotide containing a nucleic acid-like substance such as PNA
  • an association product consisting of the fluorescent probe and target particle can be formed without having to carry out special denaturation treatment even if the target particle was in the form of a double-stranded nucleic acid molecule.
  • denaturation treatment examples include denaturation by high-temperature treatment (heat denaturation) and denaturation by low salt concentration treatment.
  • heat denaturation is preferable since its effect on fluorescent substances is comparatively low and the procedure is simple.
  • nucleic acids and the like in the aforementioned sample solution are denatured by subjecting the sample solution to high-temperature treatment.
  • denaturation can be carried out by holding at a temperature of 90°C for DNA or 70°C for RNA for several seconds to about 2 minutes, since the denaturing temperature varies considerably according to the base length of the target particle and the like, the temperature is not limited thereto provided denaturation is possible at that temperature.
  • denaturation by low salt concentration treatment can be carried out by, for example, adjusting the salt concentration of the aforementioned sample solution to be sufficiently low by diluting with purified water and the like.
  • the target particles and fluorescent probe in the aforementioned sample solution are allowed to associate.
  • the target particles and fluorescent probe in the sample solution can be suitably allowed to associate by lowering the temperature of the aforementioned sample solution to a temperature that allows specific hybridization between the target particles and fluorescent probe.
  • the target particles and fluorescent probe in the sample solution can be suitably allowed to associate by raising the salt concentration of the aforementioned sample solution to a concentration that allows specific hybridization between the target particles and fluorescent probe.
  • the temperature at which two single-stranded nucleic acid molecules are able to specifically hybridize can be determined from a melting curve of an association product of the target particles and fluorescent probe.
  • a melting curve can be determined by, for example, changing the temperature of a solution containing only the target particles and fluorescent probe from a high temperature to a low temperature, and measuring optical absorbance or fluorescence intensity of the aforementioned solution.
  • the temperature range from the temperature at which the two denatured single-stranded nucleic acid molecules begin to form an association product to the temperature at which the nucleic acid molecules have nearly completely formed an association product can be taken to be the temperature at which both specifically hybridize as determined from the melting curve.
  • the concentration at which two single-stranded nucleic acid molecules specifically hybridize can be determined by similarly determining a melting curve by changing the salt concentration in the solution from a low concentration to a high concentration instead of changing the temperature.
  • the temperature at which two single-stranded nucleic acid molecules specifically hybridize can generally be substituted for the Tm value (melting temperature).
  • the Tm value of a region of a fluorescent probe that hybridizes with a target particle can be calculated from base sequence information of the fluorescent probe by using commonly used primer/probe design software and the like.
  • the temperature of the sample solution is preferably lowered comparatively slowly when forming an association product.
  • the liquid temperature of the aforementioned sample solution can be lowered at a temperature lowering rate of 0.05°C/second or higher.
  • surfactant in order to suppress non-specific hybridization, surfactant, formamide, dimethylsulfoxide or urea and the like is preferably added to the sample solution in advance. Only one type of these compounds may be added or two or more types may be added in combination. The addition of these compounds makes it possible to suppress the occurrence of non-specific hybridization in a comparatively low temperature environment.
  • step (b) the number of molecules of fluorescent particles present in the prepared sample solution is calculated. More specifically, a sample solution containing fluorescent particles and a triplet excited state quenching agent is placed in the aforementioned optical analysis device for use with the scanning molecule counting method, and by detecting and analyzing light released from the fluorescent particles according to the aforementioned procedure, the number of fluorescent particles is counted. The counted number of particles is the number of fluorescent particles contained in the measurement solution.
  • sample solutions were prepared by adding KI to Rhodamine GreenTM (AnaSpec Inc.) to final concentrations of 0 mM, 0.03 mM, 0.3 mM, 3 mM and 30 mM.
  • FCS measurement was carried out on each sample solution using the MF20 Single Molecule Fluorescence Spectroscopy System (Olympus Corp.) capable of measuring fluorescence intensity per molecule and the percentage of molecules in the triplet excited state.
  • FCS measurement conditions consisted of an excitation wavelength of 488 nm, laser intensity of 1 mW, and measuring time of 10 seconds. Measurements were carried out five times on each sample followed by calculation of the mean and standard deviation thereof.
  • FIGS. 8A and 8B The measurement results are shown in FIGS. 8A and 8B.
  • FIG. 8A is a drawing showing the percentages of Rhodamine Green molecules in the triplet excited state in each sample solution (Frac. Triplet (%)).
  • FIG. 8B is a drawing showing the amount of luminosity per molecule (count rate per particle, CPP) of Rhodamine Green in each sample solution.
  • CPP count rate per particle
  • CPP demonstrated luminosity roughly 1.3 times greater in comparison with the sample solution to which KI was not added (see FIG. 8B ) .
  • fluorescence brightness was confirmed to improve by adding triplet excited state quenching agent.
  • the sample solution having a KI concentration of 30 mM although the percentage of molecules in the triplet excited state was lower than the sample solution to which KI was not added, there was hardly any change in CPP. This is presumed to be because the frequency at which KI came in close proximity to the fluorescent dye increased due to addition of KI at a high concentration, thereby causing even those molecules in the singlet excited state to return to the ground state.
  • FCS measurements were carried out on sample solutions containing Alexa FluorTM 488 to which KI had been added to final concentrations of 0 mM, 0.03 mM, 0.3 mM, 3 mM and 30 mM in the same manner as in the case of Rhodamine GreenTM with the exception of using Alexa FluorTM 488 (Invitrogen Corp.) instead of Rhodamine GreenTM (AnaSpec Inc.).
  • FIGS. 9A and 9B The measurement results are shown in FIGS. 9A and 9B.
  • FIG. 9A is a drawing showing the percentages of Alexa Fluor 488 molecules in the triplet excited state in each sample solution.
  • FIG. 9B is a drawing showing the amount of luminosity per molecule (CPP) of Alexa Fluor 488 in each sample solution.
  • CPP luminosity per molecule
  • CPP demonstrated luminosity roughly 1.8 times greater in comparison with the sample solution to which KI was not added, thereby demonstrating an extremely potent effect (see FIG. 9B ) .
  • Cysteamine was confirmed to have quenching action by FCS measurement.
  • Cysteamine was confirmed to have quenching action on Alexa FluorTM 488 by FCS measurement.
  • sample solutions were prepared by adding cysteamine (Sigma GmbH) to Alexa FluorTM 488 (Invitrogen Corp.) to final concentrations of 0 mM, 0.3 mM, 3 mM and 30 mM.
  • FCS measurement was carried out on each sample solution using the MF20 Single Molecule Fluorescence Spectroscopy System (Olympus Corp.). FCS measurement conditions consisted of an excitation wavelength of 488 nm, laser intensity of 1 mW, and measuring time of 10 seconds. Measurements were carried out five times on each sample followed by calculation of the mean thereof.
  • FIG. 10A is a drawing showing the percentages of Alexa Fluor 488 molecules in the triplet excited state in each sample solution.
  • FIG. 10B is a drawing showing the amount of luminosity per molecule (CPP) of Alexa Fluor 488 in each sample solution.
  • CPP luminosity per molecule
  • Cysteamine was confirmed to have quenching action on TAMRATM by FCS measurement.
  • sample solutions were prepared by adding cysteamine (Sigma GmbH) to 1 nM TAMRATM (AnaSpec Inc.) to final concentrations of 0 mM, 0. 3 mM, 3 mM and 30 mM.
  • FCS measurement was carried out on each sample solution using the MF20 Single Molecule Fluorescence Spectroscopy System (Olympus Corp.). FCS measurement conditions consisted of an excitation wavelength of 543 nm, laser intensity of 0.5 mW, and measuring time of 10 seconds. Measurements were carried out five times on each sample followed by calculation of the mean thereof.
  • FIG. 11A is a drawing showing the percentages of TAMRA molecules in the triplet excited state in each sample solution.
  • FIG. 11B is a drawing showing the amount of luminosity per molecule (CPP) of TAMRA in each sample solution.
  • CPP luminosity per molecule
  • cysteamine was able to be confirmed to have quenching action on various fluorescent substances, and demonstrate an effect that improves fluorescence brightness.
  • Fluorescent particles were detected according to the scanning molecule counting method using KI for the triplet excited state quenching agent.
  • sample solutions were prepared by adding KI to 10 pM Alexa FluorTM 488 (Invitrogen Corp.) to final concentrations of 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 10 mM and 30 mM.
  • Each sample solution was measured according to the scanning molecule counting method using the MF20 Single Molecule Fluorescence Spectroscopy System (Olympus Corp.) equipped with a confocal fluorescent microscope optical system and photon counting system followed by acquisition of chronological photon count data.
  • excitation light was radiated at 1 mW using laser light having a wavelength of 488 nm, and the detecting light wavelength was set to 510 nm to 560 nm using a band pass filter.
  • the movement speed of the location of the photodetection region in the sample solutions was set to 15 mm/second, bin time was set to 10 ⁇ sec and measurement time was set to 2 seconds.
  • each sample was measured five times followed by calculation of the mean and standard deviation thereof.
  • the number of light signals detected in the chronological data was counted from the chronological photon count data acquired for each sample solution.
  • nine data points were averaged at a time, and moving average processing was repeated five times.
  • fitting was carried out on chronological data by fitting a Gaussian function according to the least squares method followed by determination of peak intensity (in the Gaussian function), peak width (full width at half maximum) and correlation coefficient.
  • peak judgment processing although only those peak signals that satisfy the following conditions: 20 ⁇ sec ⁇ peak width ⁇ 400 ⁇ sec , peak intensity > 1 photons / 10 ⁇ sec , and correlation coefficient > 0.95 were judged to be light signals corresponding to target particles, those peak signals that did not satisfy the aforementioned conditions were ignored as noise, and the number of signals judged to be light signals corresponding to target particles were counted as the "number of peaks”.
  • FIG. 12 is a drawing showing the results of counting the number of peaks of each sample solution.
  • the number of peaks increased more than in the case of the sample solution to which KI was not added, thereby confirming that the ability to detect fluorescent particles improved.
  • the number of counted peaks increased as the amount of KI added increased.
  • the number of peaks increased by 30% or more in comparison with the sample solution to which KI was not added. This is presumed to be due to the additional detection of Alexa Fluor 488 for which fluorescence brightness was enhanced by KI.
  • Fluorescent particles were detected according to the scanning molecule counting method using cysteamine for the triplet excited state quenching agent.
  • sample solutions were prepared by adding cysteamine to 10 pM Alexa FluorTM 488 (Invitrogen Corp.) to final concentrations of 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 10 mM and 30 mM.
  • Each sample solution was measured according to the scanning molecule counting method in the same manner as Example 1 followed by acquisition of chronological photon count data and counting of the number of fluorescent particles.
  • FIG. 13 is a drawing showing the results of counting the number of peaks of each sample solution.
  • the number of counted peaks increased as the amount of cysteamine added increased.
  • the number of peaks increased by 20% or more in comparison with the sample solution to which KI was not added.
  • sample solutions were prepared by adding cysteamine to 10 pM TAMRATM (AnaSpec Inc.) to final concentrations of 0 mM, 0. 1 mM, 0.3 mM, 3 mM, 10 mM and 30 mM.
  • Each sample solution was measured according to the scanning molecule counting method in the same manner as Example 1 with the exception of radiating excitation light at 0 . 5 mW using laser light having a wavelength of 543 nm and setting the detecting light wavelength to 560 nm to 620 nm using a band pass filter, followed by acquisition of times series photon count data and counting the number of fluorescent particles.
  • FIG. 14 is a drawing showing the results of counting the number of peaks of each sample solution.
  • the number of counted peaks increased as the amount of cysteamine added increased.
  • the number of peaks increased by 10% or more in comparison with the sample solution to which KI was not added.
  • sample solutions were prepared by adding KI to 1 pM or 100 pM Alexa FluorTM 488 (Invitrogen Corp.) to final concentrations of 0 mM, 0.1 mM, 0.3 mM, 1 mM, 3 mM, 10 mM and 30 mM.
  • Each sample solution was measured according to the scanning molecule counting method in the same manner as Example 1 with the exception of using a measurement time of 20 seconds for sample solutions having an Alexa Fluor 488 concentration of 1 pM and using a measurement time of 2 seconds for sample solutions having an Alexa Fluor 488 concentration of 100 pM, followed by acquisition of chronological photon count data and counting the number of fluorescent particles.
  • FIG. 15 is a drawing showing the results of counting the number of peaks of each sample solution having an Alexa Fluor 488 concentration of 1 pM
  • FIG. 16 is a drawing showing the results of counting the number of peaks of each sample solution having an Alexa Fluor 488 concentration of 100 pM.
  • the ability to detect peaks (ability to detect fluorescent particles) during measurement according to the scanning molecule counting method improved as a result of adding KI in the same manner as Example 1 (case of sample solution having an Alexa Fluor 488 concentration of 10 pM).
  • the concentration at which KI demonstrates quenching action was determined to not be significantly affected by the concentration of fluorescent particles, and fluorescent particles were determined to be able to be detected with higher sensitivity by adding KI to the sample solutions at a final concentration of 0.1 mM to 20 mM.
  • the concentration of target particles only present at an extremely low concentration in a sample solution can be measured according to a scanning molecule counting method with extremely high sensitivity. Consequently, the method for detecting a fluorescent particle in a mode of the present invention can be used in fields such as analysis and testing of samples such as clinical specimens in which the concentration of a substance to be analyzed is extremely low.

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CN103718023A (zh) 2014-04-09
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EP2743684A1 (en) 2014-06-18
US20140131593A1 (en) 2014-05-15
CN103718023B (zh) 2017-03-15
JPWO2013024637A1 (ja) 2015-03-05
WO2013024637A1 (ja) 2013-02-21

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